An increasing body of evidence suggests that alcohol affects gene expression in the brain more than had been previously thought. This is particularly tue during periods of chronic ethanol treatment, when gene expression in the central nervous system is subject to the neurochemical changes associated with the development of tolerance and dependence. The proposed project will examine the evolution of ethanol addiction as a function of the responsiveness of the initial product of gene expression, messenger RNA (mRNA). The molecular cloning techniques of "differential colony hybridization" and "subtracted library hybridization" will be used to identify and characterize complementary DNA (cDNA) clones of rat brain mRNA altered (induced or repressed) as a result of ethanol dependence an withdrawal. Rats will be rendered ethanol-dependent by repeated intragastric administration of high doses of ethanol over a 5 day period. Complementary DNA libraries will be constructed using mRNA isolated from the brains of control and ethanol-dependent rats as well as dependent rats withdrawn from ethanol. These cDNA libraries will be subjected to recombinant hybridization techniques which will reveal cDNA clones for mRNA which undergo up-or down-regulation in ethanol dependence an withdrawal. Differentially-expressed clones will be characterized based upon nucleic acid sequence and according to the size, tissue distribution and abundance of each complementary mRNA. A time-course analysis will be performed to monitor ethanol-induced changes of these mRNA, and selected clones will be used to localize mRNA to ethanol-responsive sites in rat brain. The experimental design of this proposal will provide an unbiased approach for the discovery of cDNA clones that can be used to study gene expression in the central nervous system during the transition from acute ethanol intake through ethanol dependence and withdrawal. Selected differentially- expressed cDNA clones will eventually be used as molecular probes for expansion of the data base of molecular correlates found in ethanol addiction. Future studies exploring the mechanisms of ethanol-induced changes in gene expression (i.e., transcription), neuro-anatomical loci for the molecular basis of addiction (i.e., in situ hybridization) and studies of the genetic linkages and restriction fragment length polymorphisms in alcoholism will also be facilitated by these cDNA clones.